Tip:
Highlight text to annotate it
X
Professor Kevin Ahern: Friday has arrived!
Is everybody aware of that?
What Friday, what's Friday, right?
Alright, we've got material to get through.
Exams are starting to loom big in our event horizon,
as it were.
The first exam of the course will be a week from Monday.
Oh joy, eh?
So I'll say more about that next week.
We'll have a review session at some point,
and I hope to have information about that for you soon.
Today I'm going to finish up with hemoglobin,
and I hope I convinced you last time
that hemoglobin was a pretty amazing protein,
and that there are many structural aspects to hemoglobin
that allow it to do what it does.
And understanding something about those structural elements,
or the structural considerations in hemoglobin,
allow you to begin to better understand
how it is that enzymes function.
I want to emphasis that hemoglobin is not an enzyme.
Enzymes catalyze reactions, and there's no reaction being catalyzed by
hemoglobin.
It's sole function is carrying oxygen
and dropping it off where it's needed.
So it's not catalyzing a reaction,
but in some ways the structural considerations
that we have for hemoglobin carry over into enzymes,
and we'll see how that happens very shortly.
Well there's one last thing about hemoglobin
that I want to mention-
as if it wasn't remarkable enough as it was-
to think about,
and that actually relates to sickle cell anemia.
So sickle cell anemia is a genetic disorder.
It can actually arise in a variety of ways,
a variety of different mutations in hemoglobin
can give rise to sickle cell anemia.
The blood cell that is shown here in sort of a yellowish color
is a blood cell that would be found in an individual
that has sickle cell anemia.
And this sickle shape arises as a result of
low oxygen concentration.
Where would a blood cell encounter low oxygen concentration?
That would happen, of course, in any tissue
where the tissue is actively respiring,
and generally this would occur let's say in muscle tissue,
and specifically it would occur in the capillaries
because this is the place where there's interaction between
the hemoglobin component and the oxygen
and of course the cells that are taking up that oxygen.
This is exactly the wrong place
for this blood cell to assume this form
because this sickle shape that you see right here
isn't nice and rounded and smooth.
It gets stuck in capillaries.
It gets stuck in capillaries.
Well, when it gets stuck in capillaries,
it plugs up capillaries,
and since capillaries are primary places
where oxygen is being dropped off,
those muscle cells that were relying on hemoglobin for oxygen
are suddenly starved for oxygen.
Not surprisingly,
sickle cell anemia is a debilitating disease.
It causes a person to have problems with heavy exertion.
The disease itself can be fatal,
as you can imagine,
if you were unable to supply oxygen as needed
for a period of time.
It's called an anemia because anemias are diseases
that relate to the shortage of red blood cells.
And what happens with this
is once this guy has assumed this sickle shape,
the body looks at it and says,
'oh we've got a damaged blood cell,
'we take it out of action.'
So it takes out of action blood cells that would otherwise
be normal and functional,
and as a consequence, since a lot of these-
especially during periods of heavy exertion-
appear in this form,
the person's blood count is always going to be low.
They are always scrambling
to produce enough red blood cells to satisfy demand.
So it's a very debilitating disease.
The reason the sickle shape forms
is because the hemoglobin within this blood cell
will actually form polymers.
That is they form long, long cancatomers of hemoglobin units,
one after the other after the other,
and change the shape of the hemoglobin.
In this normal red blood cell that we can see on the right,
hemoglobins are individual units, they are not polymers.
So these polymers cause this sickling shape to happen,
and the polymers happen because of some very simple mutations
that occur in the coding for hemoglobin,
that is, the 'globin' part of the hemoglobin.
Well that's, as we can imagine, pretty nasty.
Here you can see some of these polymers,
here a little more closely,
and that's exactly what's happening inside
or associated with this portion of the red blood cell,
and one of the questions people ask
with almost any disease is,
'Why does this disease persist in the human population?'
This is a genetic disease and if people are carrying it,
there must be some reason why it hasn't been eliminated
from the human population by the process of selection.
And it turns out that when researchers examined
the incidence of sickle cell anemia around the world,
in terms of numbers, and they compared that
to a variety of other diseases around the world,
what they found was that the disease of malaria
overlaid very similarly the map of sickle cell anemia.
Sickle cell anemia can strike,
or it doesn't occur just in people in sub Saharan Africa,
but it's more predominant in areas of the tropics
that you see shown here in yellow,
sorry, shown here in this sort of reddish.
So it can occur anywhere, it's not just in that one place,
but the high incidence of it here suggests
that there might be some selective advantage
to having at least the sickle cell anemia gene,
and it turns out that there is.
The sickle cell anemia gene is a recessive trait.
So it takes two recessives
to give the full sickle cell anemia phenotype.
That phenotype turns out to be the one
that is very problematic.
It is very, can be deadly, can be very debilitating.
But people who are heterozygous,
who have one normal allele and one mutant allele,
that is sickle cell anemia,
appear to have a selective advantage,
particularly when they're young.
The incidence of death due to malaria
for people who are heterozygous for sickle cell anemia
is lower.
So there is some selective advantage.
The organism that infects people and causes malaria
is dependent upon oxygen,
and so it may well be
that what we see as a problem is actually an advantage
in terms of what this organism that's requiring oxygen
is actually needing and doing.
So there may be some advantage to having the allele,
at least in heterozygous form.
Yes?
To be honest with you,
I probably can't and shouldn't answer that question,
and the reason is that there are people
who sue you for medical purposes
and so much as I would like to give you my opinion on that,
I probably shouldn't do that,
and since people all over do watch these videos-
I apologize, I generally will avoid that sort of thing.
But if you want to come by and talk
I would be happy to talk to you about that.
Yes, question back there?
You know I don't know David, the answer to that question.
I don't know which one corresponds to that.
I'm sorry.
So that's hemoglobin, an absolutely fascinating protein.
We're going to turn our attention now
to some other absolutely fascinating proteins,
in their own way, and these are enzymes.
Enzymes, of course, are proteins that catalyze reactions,
and enzymes are incredible.
Hemoglobin, I told you was incredible,
and when I use the term incredible
I hope I have some credibility, ha ha, for you with respect.
Thank you,
[students laughing]
with respect to that.
You're laughing already, right?
Maybe what I should do instead of this
is do our song early instead of do our song late
since you're already in a laughing mode.
Would that work?
Okay, let's do the song early.
So the song actually relates to hemoglobin,
so maybe it's appropriate we do it at this time,
and it's to the tune of an old song
that Coca-Cola used to sell Coca-Cola with.
It's called, "It's the Real Thing."
You guys know the song?
"I'd like to teach the world to sing in perfect harmony."
You can try it, alright?
Try it with me.
It's called "The Bloody Things."
[Professor Kevin Ahern and students singing]
I'm gonna put some oxygens
beside my porphyrin rings.
To nudge the irons up a notch
and yank on histidines.
The globins' shapes will change a bit,
oh what a sight to see.
The way they bind to oxygen
"co-op-er-AH-tive-ly."
And as I exit from the lungs
to swim in the bloodstream,
metabolizing cells they all
express their needs to me.
To them I give up oxygen
and change from R to T,
while my amines, they hang onto
the protons readily.
But that's not all the tricks I know,
there's more that's up my sleeve.
Like gaps between "sub-U-nits" that
hold 2,3-BPG.
When near met-a-bo-LI-zing cells,
I bind things that diffuse.
The protons and bicarbonates
from lowly cee oh twos.
That's the way it is
when your cells are at play.
Go say hi hip hooray
for the bloody things.
Professor Kevin Ahern: Alright,
so we've got our song out of our system, that's good.
Alright, coming back to enzymes.
Enzymes are, as I said, remarkable proteins,
and I just want to just give you a little bit
of a glimpse of that.
We take them for granted, okay?
So let's look at the rate of enhancement
of a variety of enzymatic reactions.
There's a lot of information there.
But let's look to see some of these different reactions
that are catalyzed.
So here's an enzyme called OMP decarboxylase.
We'll talk about it next term
when we talk about how nucleotides are made.
This enzyme catalyzed a reaction
that, if we were to look at the same reaction
in the absence of the enzyme,
the half-life of that reaction would be 78 million years.
78 million years.
When we look at what this enzyme does
when we add it to the solution,
the enzyme catalyzes the reaction at the rate
of 39 molecules of product per molecule of enzyme per second.
Well if we do the math and we calculate all this,
what we discover is that this is an enhancement
that is how many times faster it's actually doing it?
The enhancement is 1.4 x 10 to the 17th.
That is 140 quadrillion times faster.
A chemical catalyst like platinum is pretty good
if it speeds things up 100 to 1000 fold,
and here's something that's speeding up something
140 quadrillion times faster than the same reaction
in the absence of the enzyme.
That's pretty hard to get your head around.
There are other things about enzymes
that are hard to get your head around.
We'll talk more about this column over here later,
but I want to show you something
about the last enzyme on the list, carbonic anhydrase.
Carbonic anhydrase catalyzed a reaction
that normally doesn't take that long to go through,
but look at how many molecules of product it makes
per molecule of enzyme per second.
What does that mean?
This says that this enzyme will produce
one million molecules of product per molecule of enzyme
every second.
I take one enzyme molecule, I take the things it catalyzes,
and one million of them will be converted
into product per second.
That's pretty hard to get your head around.
That's pretty hard to get your head around.
We think about computers operating really fast,
et cetera et cetera.
Here's a molecular process that's occurring like a computer.
A million molecules of product per second
for every single enzyme that's in that solution.
Well this is so hard for us to get our heads around,
I like to use this as an example of the fact
that, when we think about things at the nanoscopic scale,
we're really thinking about a different kind of world
than what we live in.
There is nothing out here in the world that we live in
that we can experience that gives us
a million things per second.
Enzymes are really remarkable.
Because they are so remarkable
and because they are so much more remarkable
than chemical catalyst,
we have to understand how it is
that enzymes accomplish the magic that they accomplish.
To do that we need to give some considerations
to what's known as free energy.
You guys took free energy
when you took your freshmen chemistry
probably in your last term,
and you probably went, 'Oh my god,'
and you're probably sitting here now thinking the same thing,
because now he's going to make us do more calculations.
Well for what I'm going to talk about here,
we're not going to really do much in the way of calculations.
We will do some calculations related to free energy later
when we talk about metabolism.
Right now our consideration of free energy
is simply related to what's here
and how free energy plays into what enzymes do.
So free energy is energy that is available
for useful purposes.
Useful purposes.
So we understand the free energy of a reaction.
Basically we can understand
if a reaction is feasible or not feasible.
Now I'm going to give you a little heads up here,
and you probably learned this in freshmen chemistry
but you didn't remember it,
enzymes and catalysts, they're both catalysts,
do not change the overall free energy of a reaction.
They do not change the overall free energy of a reaction.
So all the changes that happen
do not relate to the overall free energy-
that's important to understand.
Well I think you probably learned in freshmen chemistry
that the delta G,
which is the change in free energy for a given reaction,
tells us, or allows us, to predict
what the direction of a reaction will go.
If the delta G for a reaction is negative,
that is less than zero,
the reaction will go forwards as it's written.
If the delta G for a reaction is equal to zero,
the reaction is at equilibrium.
If the delta G for a reaction is positive,
the reaction will go backwards as written.
Three very simple rules.
One not so simple rule, notice the last point there,
"equilibrium does not mean equal concentration
"of reactants and products."
The most common misconception that I deal with,
that seniors in science would still think this
is remarkable to me.
Don't make that assumption.
Don't fall into that trap.
Equilibrium does not mean equal concentrations
of reactants and products.
Now, those simple rules right there, as I said,
allow us to predict the direction of a reaction.
If we look at the equation
that allows us to calculate the free energy for a reaction,
we see that that equation is right here.
You notice in that equation that there's another term
that looks like delta G,
and it's labeled as delta G zero prime.
That's a big zero and that's a prime over there.
It says that the overall free energy for a reaction,
which is the delta G, is equal to the delta G zero prime,
plus R, the gas constant R, times the temperature,
times the natural log of the concentration
of the products over the reactants.
That's what it says.
I'm going to explain that to you, but that's what it says.
What that says is that the delta G is equal to
a different kind of delta G plus
this term times the concentration of products and reactants.
This equation looks very foreign
but this equation is almost identical to Henderson Hasselbach.
PH, which is something that we can measure,
is equal to a constant, which is pKa,
this guy right here turns out to be a constant
for a given reaction also, plus the log,
well this is a natural log,
of the concentration of salt over acid.
It's the same equation, it's the same basic idea
that you learned in Henderson Hassabach
that you work with in this equation.
We're not going to do equations at this point.
We're not going to do calculations at this point,
so I'm not going to go through that any further.
I will come and talk about this more
when we discuss it later-
when we discuss metabolism later.
What I do want to introduce however
is the concept of energy with respect to chemical reactions.
So let's take what I've just introduced about energy
and think about how enzymes work.
No that's not the one I want, the one I want is the next one.
This is a really dandy figure.
It actually shows us everything about how enzymes work.
Everything.
Let's look at it.
I'm going to describe it to you in mathematical terms,
and then I'm going to give you
a more real-world kind of a description.
Notice that we are plotting in this case
the free energy versus the reaction progress.
When we look at this graph
we see the free energy of the material to start with,
and we see the free energy of the product and the delta G,
which is the change in energy,
is the difference between this right here and this down here.
That is the difference between the energy of the substrate
and the energy of the product.
You'll notice that we see two things going on here.
We see in the sold line a reaction that is uncatalyzed-
no enzyme, no catalyst.
We see that during the course of the reaction,
the formation of what we call the transition state
causes the free energy to increase to a significant amount.
Once it gets over that hump,
it goes down here and makes product.
This might be two molecules bumping into each other
with the right energy,
this might be two molecules bumping into each other
with the right energy and the right orientation,
and this amount of energy is necessary
to get the reaction started.
We see that if we had an enzyme we see a different situation.
We start at the same energy level,
but instead of having to go up this hump right here
we go up a much lower hump
and we produce the same product that has the same energy.
The energy change for the enzyme catalyzed reaction
is identical to the energy change
for the non-enzyme catalyzed reaction.
The enzyme didn't change the overall free energy,
but during that reaction, there were some things that changed.
The real world analogy I have to this
is I always like to have the class envision
the following scenario.
Imagine, if you will,
that we are at about 300 feet above sea level
right here in Corvallis.
Sea level of course is zero feet
because that's what sea level is.
In theory, we should be able to take a giant ball bearing
that's the size of about half the size of this room
and push it in the direction of either the Pacific Ocean
or the Atlantic Ocean and it would get there,
because we're higher than what those oceans are.
The overall change is going to be 300 feet
and we know that things roll downhill.
But we also know, as we think about it,
that it's not a straight going down the slope
to either the Pacific or the Atlantic Ocean.
We know that along the way there are hills
and so forth as we go over there.
So if you wanted to make sure
that thing got to the pacific ocean, you would say,
'Well let's take this ball bearing as a class,
'and we're going to push this ball bearing,'
and were going to assume we don't have trees
and so forth in the way.
If we have enough clear cutting,
we'll have that happen I suppose.
But if we take this ball bearing and we push it
up to the top of Mary's Peak,
that the tallest peak in the area,
we know that if it gets up there
it will go down and up and down and down and down,
and finally it will arrive at the ocean, and that will work.
As long as obviously nothing is blocking its path.
What I just described to you
is what a non-enzymatic reaction does.
It takes it to the highest place
where there is no turning back
and then it goes down that path,
ultimately down to the ocean.
What enzymes do is they say,
'Why should I go to the top of Mary's peak?
'That's going to be the hardest place for me
'to push the ball bearing.
'All I need to do is push it up to the pass
'and get it to that point.'
I push it to the pass, the highest pass that I need to get to
and it will do the same thing.
Enzymes are therefore lowering the energy necessary
to get to that transition state,
and by doing so they enable many, many more molecules to get
to that transition state.
This transition state, as I noted,
is essential for the reaction to proceed.
OK, questions about that?
Delta G is exactly the same for both.
Delta G does not change for an enzymatic reaction.
And that means that, therefore,
an enzyme will not change the equilibrium for a reaction.
It will not change the equilibrium.
It's completely unchanged.
Now, what we see here is that what an enzyme is doing
is it's transiently changing the free energy
of the intermediate.
It's transiently doing that.
And that turns out to be a really interesting phenomenon,
OK, a really interesting phenomenon.
Alright, how does the enzyme manage to do that?
And how does it manage to do that so much better
than a catalyst?
A catalysts will also lower the transition energy for
a reaction, that's how a catalyst works as well,
a non-enzyme reaction, alright.
But it doesn't do it as well as an enzyme does.
What is so magical about an enzyme?
Well, when people used to study biochemistry,
they used to think there were all kinds of magical things
about biochemistry because things happened inside of cells
that really didn't seem to make any sense outside of cells.
Well, we know of course now,
that the chemical reactions that occur inside of cells
are no different than those outside of cells.
The delta G's don't change.
The rates may change, and we have to understand
how it is that the rates of those reactions change.
Well, I said that a non-chemical catalyst like platinum
will reduce that transition state energy a little bit,
but nothing like what an enzyme will do.
So how does an enzyme do something different
from what a chemical catalyst like platinum does?
Well the answer turns out to be something
that you have already studied and learned.
Enzymes are flexible.
Enzymes are flexible.
It's the flexibility of enzymes
that allows the enzymes to perform the magic that it does.
Flexibility, okay?
We'll talk later about an enzyme called hexokinase.
It's one I usually like to give as an example.
Hexokinase catalyzes the first reaction
in the metabolism of glucose.
To do so what hexokinase has to do
is it has to take glucose,
and it has to take some ATP,
and it has to transfer a phosphate from the ATP
onto glucose.
If I wait for that to happen inside of a test tube
by simply mixing ATP and glucose,
it will take a heck of a long time to happen
because the ATP, over here has to bump into glucose
in exactly the right orientation,
and then separate in order for that process to occur.
Alright, and so I wait a long time-
there's going to be many times they're going to hit,
they're going to be in the wrong orientation,
the phosphates in the wrong place,
they're upside down,
they're backwards, or whatever.
So many, many interactions that happened
in the absence of a catalyst are non-productive.
The enzyme does two things to ensure
that this reaction occurs.
First, the enzyme has, on it,
a specific binding site for glucose.
It also has a specific binding site for ATP.
And schematically, if we were to look at them,
they look kind of like this.
Okay?
Enzymes as I said are flexible.
They orient things so that the glucose
is positioned perfectly up here on the top
and the ATP is positioned perfectly here on the bottom,
such that the binding of the two causes a small shape change.
You've seen small shape changes happen in hemoglobin, right?
The small shape change that happens is this.
The phosphate is placed immediately next to the glucose.
It doesn't have to accidentally get there.
And then, as the phosphate jumps from one to the other,
another shape change happens.
The jaws open.
When the jaws open, they let go of the substrate, and guess what?
You've done exactly what you would be waiting
a long time in a test tube to have happen.
Enzymes have specific binding sites
and enzymes have specific 3D-orientations
that allow the substrates-
and by the way the substrate is what
the enzyme is catalyzing its reaction on-
they allow the substrates to be in perfect position to react.
Chemical catalysts cannot do that.
They don't have flexibility, they don't have binding sites.
Yes sir?
How does the substrate binding into the enzyme
cause the enzyme to bend?
By the very same mechanism
that the binding of oxygen inside of hemoglobin
caused hemoglobin to change shape slightly.
We'll see inside of enzymes
that sometimes the bends are actually pretty big.
And your question is how does that happen?
Imagine, if you will, that when you bind a sub-straight,
what are you changing inside of that enzyme?
What is going to cause a substrate to bind?
Various types of bonding.
So you could have hydrogen bonding,
you could have hydrophobic bonding, and so forth.
And as you do that you're changing the electronic environment
of the enzyme itself.
As you change the electronic environment,
when you think of electronics what do you think of?
Charge, and when you start thinking about how charges change,
you start thinking about how those interactions
that stabilize the enzyme change,
and now you start thinking
that the enzyme is going to adapt to that.
That's exactly what happened in hemoglobin,
and that's exactly what's happening inside of an enzyme.
Does that make sense?
Pretty cool stuff.
Other questions?
OK, now, I'm going to spend,
actually probably next week, some time talking about
mechanisms of catalysis.
So I've just given you a little taste of the mechanisms
of catalysis.
What I'm going to spend some time on now,
and probably for the next couple of lectures,
is I'm going to spend some time talking about
what we call the kinetics of an enzymatic reaction.
Kinetics relating to speed, movement.
That's what I'm going to talk about here.
And what you're going to see as I talk about this
is you're going to see some things
that look kind of like what hemoglobin was doing.
Kind of like what hemoglobin was doing.
Here is an example of the type of kinetics
that we study with enzymes.
One of the things that we're interested in studying in
enzymes is the reaction velocity.
Because you've already seen that enzymes
can be pretty remarkable in how fast they can do things.
We want to understand how that velocity of reaction
can be studied.
Well if I say velocity to you,
thinking about driving around in your car,
or riding on your bicycle, or jogging,
or walking, or whatever,
we think about it's a rate.
It's a distance divided by time.
50 miles per hour, 20 miles per hour,
three steps per second.
There's something having to do with a rate.
When we talk about an enzyme's velocity we're not talking about
movement, we're talking about reaction.
So what do enzymes do, they catalyze reactions.
They take substrates and they convert them into products.
So for an enzymatic velocity
we need to understand the rate of formation of product.
The rate of formation of product.
Enzyme velocity is equal to
the concentration of product over time.
The concentration of product over time.
That's pretty straight forward.
That is expressed as molarity per second.
Molarity per minute, millimolarity per second,
millimolarity per minute.
That's what the velocity component is.
How would I measure velocity?
Well maybe I should tell you more about
how I would produce this plot right here.
Velocity is one thing I'm interested in.
If you look at this plot,
what you see is that the velocity goes up
as the substrate concentration goes up.
That's not totally surprising.
Let's imagine I've got an enzyme sitting over here.
And this enzyme is sitting here waiting for
a substrate to bind to it
and then to be converted into product.
If the enzyme is sitting here waiting for a period of time
before the substrate gets close to it,
and before the substrate binds to it,
the enzyme is wasting its time.
If I add and increase the substrate concentration
so that I've got 10 times as much substrate
as I had in the first situation,
then I would have a 10 times more likelihood
that the enzyme and the substrate would bump into each other
and the enzyme could catalyze a reaction.
Intuitively it means that the more substrate I have there,
the more likely the enzyme is going to bind to it
and the more likely a product will be formed.
When I do that, I see that as I increase
the substrate concentration this plot occurs.
That looks kind of like what we saw with myoglobin,
binding to oxygen.
We saw what we call, I didn't give it a name at the time
but I'll give it a name here, we saw a hyperbolic plot.
Myoglobin's binding plot for oxygen is hyperbolic,
and this plot that I just described to you,
which is called velocity versus substrate concentration,
this is also a hyperbolic plot.
Well you see there's something on this graph
that says maximum velocity.
It says that this is going to reach a maximum
and then it's not going to go any faster.
Why is that?
Well I like to compare enzymes to factories.
A factory that makes automobiles takes substrates,
which are just simply parts for assembling a car,
it takes substrates, it takes the parts for a car,
and workers work on assembling those automobiles
and when they're done they have
a finished product, an automobile.
A factory has a given capacity.
If you give a factory an excess of parts,
if you give it way more parts than it needs,
it still can't produce them any faster
because adding more and more parts to that total
of what they've got doesn't change how fast the workers
can put everything together to make that automobile.
If I'm short of parts
and the supplier is kind of dribbling them to me
and I'm getting them at a very slow rate,
that really could affect me at the low end where,
well we're twiddling our thumbs
waiting for parts to get here, right?
But at the high end I run into a maximum.
This is what enzymes do.
Enzymes run into maximums
because we can increase the substrate concentration
as much as we want, but once we get it to the point,
where as soon as a reaction is done
another substrate is there,
we can't make it go any faster than that.
So, enzymatic reactions will have a maximum velocity.
Everybody with me?
Maximum velocity turns out not
to be a characteristic of an enzyme.
That may seem a little cockeyed to tell you that,
but it means it's not a characteristic of an enzyme.
When I say it's not a characteristic what does that mean?
It means that the velocity of the reaction
is not something inherent to the enzyme.
It's inherent to how many enzymes I have.
If I take general motors and I'm assembling an automobile
and I have one factory that produces GMC trucks
the number of GMC trucks that's going to come out
is going to be the maximum velocity of that GMC truck factory.
If I make another factory that makes trucks,
and it has the same rate as the first factory,
my velocity of making trucks is going to double.
I can produce twice as many trucks
because I've got two factories that are working on that.
As long as they have enough stuff to use,
they will produce twice as many trucks.
If I make three factories I'll have three times as many.
Enzymes are the same way.
If I have a given velocity for a given amount of enzyme
and I double the amount of enzyme
I'm going to double the amount of the maximum velocity.
So maximum velocity, if I'm going to talk about it,
really has to be related to,
"how much enzyme did you use Ahern?"
You've got to think about that.
So maximum velocity is not inherent to an enzyme,
it's inherent to a concentration of enzyme.
It's for that reason maximum velocity is not used.
It's not any good for comparing enzymes.
I can't compare the velocity of one enzyme with another enzyme
because, again, it's dependent
upon the concentration of the enzyme.
Well I would like to be able to compare enzymes,
so what can I do to compare them?
In order to do that, what I need to do is,
I need to take into consideration
the concentration of enzymes.
Alright?
I'm going to give you a term that
allows you to understand that, okay?
And that is, where did it go?
I've lost it, but I'll tell you what it is.
It's called Kcat.
Kcat is related to the maximum velocity,
but it takes the enzyme concentration into consideration.
Kcat is equal to the maximum velocity-
By the way, maximum velocity is also abbreviated Vmax-
Vmax is maximum velocity.
Kcat is equal to Vmax divided by
the concentration of enzyme that you used.
Now, you've taken into consideration the enzyme concentration.
And because of that, you've reached something that is
a property of a given enzyme.
Kcat can be compared between enzymes.
When I showed you the table earlier today,
what I was showing you in comparing those enzymes
were Kcats.
That's Kcat right there.
The units on Kcat are per second.
That's the units.
What it translates to is
molecules of product per molecule of enzyme per second.
So something that has a Kcat of one million,
means that each molecule of enzyme is catalyzing the formation
of one million molecules of product per second.
Well, I can say that this enzyme down here,
carbonic anhydrase, is working a heck of a lot faster
than any of these other enzymes
because I've taken the concentration of enzymes
out of the equation.
Make sense?
Everybody understand Kcat
and the difference between Kcat and Vmax?
No?
If you don't, this is a good time to ask.
Yes?
Kcat is not a molarity.
Kcat is a rate,
and it's a rate of product per enzyme per second.
Product per enzyme per second.
Vmax was a molarity.
Vmax was a molarity per second, right?
And that was molarity of product per second,
or per minute or whatever.
It's a very good question that she's asked here.
Well what happens to the molarity term?
Well, what did we do?
We divided concentration of product
by the concentration of the enzyme,
and the concentrations disappeared-
we were simply left with a number.
And that's why it's number of molecules of product
per molecule of enzyme, per time, per second.
Ok?
So that's what happened to the molarity term.
We took it out of the equation.
Yes?
The units of Kcat are just per second,
or per minute, or whatever we define as time.
OK, so Kcat turns out to be a really useful term
for us to understand enzymes.
Alright, now, since I'm on the topic-
I'm jumping around a little bit here today-
Since I'm on the topic
I'm going to also introduce another term to you,
I'm going to say more about these next time,
and that is a term known as KM.
This shows you the graph I was talking about earlier,
but it shows you in a little bit more detail,
and I also wanted to do two things here.
One is to come back and tell you
about how we make such a plot,
and number two,
what's useful and informative for us out of that plot.
Please note that we always calculate Vmax.
We have to calculate Vmax in order to calculate Kcat.
Kcat doesn't just jump out at us.
We've got to calculate Kcat from our Vmax.
So we have to first determine Vmax
and then divide by the concentration of enzyme that we used.
Well first, let's talk about how we make this plot.
If I want to make this plot, I'm going to do an experiment,
and I'm going to do an enzyme kinetics experiment.
I'm going to take, let's say, 20 different tubes
of reaction that I'm going to study.
All the tubes are going to have enzyme.
All the tubes are going to have buffer.
And to each tube I'm going to add
a different amount of substrate.
Low concentration of substrate,
high concentration of substrate,
and varying concentrations of substrate in between.
I let the reaction go for a fixed period of time,
and if I do this right
I try to do this for a very short period of time.
The reason I do it for a short period of time
is that I don't want my reaction to come to equilibrium.
What happens at equilibrium?
Here's a good question for those of you
who know something about equilibrium.
What happens at equilibrium?
The forward reaction equals the reverse reaction.
I don't want to study the reverse reaction,
I'm interested in the velocity of this reaction.
I want to see what's going forwards.
I want to study this before I get to equilibrium
because if I get to equilibrium
I'm not going to see anything happen.
So I want to study this very early.
So I usually study this for a very short period of time,
before product has much of a chance to accumulate.
If product accumulates too much,
then I'm going to start studying the reverse reaction,
and that's not going to tell me anything.
Make sense?
Ok, now,
you've learned how it is that we make a plot like this.
This graph has one other thing on it
that we need to understand.
That's what I'm going to finish with for today
and I'll say more about it next time.
We've learned a very important parameter about enzymes,
that was Kcat.
This thing I'm getting ready to show you
is another important parameter about enzymes,
and it's called KM.
It's also called the "Michaelis constant".
What KM tells us that's very valuable
is the enzyme's affinity for its substrate.
What is affinity?
Affinity is what you have for your significant other.
That's a joke.
[laughter]
Tight binding,
all the various things that you can think of here, right?
All the metaphors you want to have.
If you really like your significant other
you may have more affinity
than if your significant other is not so significant, right?
You understand what I'm talking about.
I'm going to tell you the answer
and then I'm going to tell you how we get to this next time.
The answer to this is
something that has high affinity has a low KM.
Something that has low affinity has a high KM.
We measure affinity by measuring the concentration,
the concentration,
that it takes for a reaction to get to half of Vmax.
We measure the concentration of substrate
that it takes to get a reaction to half of Vmax.
Why don't we measure
the concentration it takes to get to Vmax?
There is no such thing.
This concentration will get me to Vmax,
this concentration will get me to Vmax,
this concentration over here on the wall will get me to Vmax.
There's no one concentration.
But there is a specific concentration
that will get me to half of Vmax,
and that's something I can measure very readily,
and this tells me something
about the enzyme's affinity for its substrates.
We'll pick up at that point next time.